Method of heat fractionating ldh into isoenzyme components

ABSTRACT

A method of effectively heat fractionating lactic acid dehydrogenase-containing samples into their isoenzyme components to determine abnormal LDH activity for diagnostic purposes is disclosed which relates to a rapid heating step in the presence of a buffered solution having a certain concentration and carried out at a specified pH range.

United States Patent [1 1 Rush et al.

[ 1 Jan. 21, 1975 METHOD OF HEAT FRACTIONATING LDH INTO ISOENZYMECOMPONENTS [75] Inventors: Robert L. Rush, Spring Valley; Anne C. Delea,Yonkers; Milos Stastny, Ossining; Edward D. Lash, Tarrytown, all of N.Y.

[73] Assignee: Technicon Instruments Corporation,

Tarrytown, N.Y.

[22] Filed: June 9, 1972 [21] Appl. No.: 261,500

[52] US. Cl. 195/1035 R, 195/66 R [51] Int. Cl G01n 31/14, GOln 33/16[58] Field of Search 195/1035 R [56] References Cited OTHER PUBLICATIONSStrandjord et al., J. Am. Med. Ass0cn.,

182(1l):l099-l102, (1962).

Don et al., Enzymologia," 36(6):353-370, (1969).

Vesell et al., Proc. Nat. Acad. Sci, 56zl3l7-l324, (1966).

Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or FirmMorgan,Finnegan, Durham & Pine 1 ABSTRACT 24 Claims, N0 Drawings METHOD OF HEATFRACTIONATING LDH INTO ISOENZYME COMPONENTS BACKGROUND OF THE INVENTIONThis invention relates generally to a heat fractionation techniqueapplied to serum samples to fractionate the isoenzymes which are foundin the lactic acid dehydrogenase (LDH) component. More particularly, itrelates to a rapid fractionation of the serum LDH by heating the serumsample for a short period of time at a pH of from 7 to in the presenceof a buffer solution at a concentration which permits substantialinactivation of certain or specific isoenzyme molecules.

LDH is an enzyme found in human tissue, particularly abundant in kidney,cardiac, hepatic and muscular tissues. Since the LDH level remainsrelatively constant in normal serum samples, its elevation is indicativeof tissue disfunction or disease.

The determination of LDH activity in a blood sample relies on the use oflactate as the substrate in alkaline solution which causes theconcurrent reduction of nicotinamide adenine dinucleotide (NAD) to itsreduced form, i.e., NADH. This latter conversion is measuredphotometrically. The concentration of enzyme (LDH) is directlyproportional to the increase in NADH concentration.

Procedurally, the above sample to be assayed for LDH activity isgenerally heated at 37C. at a pH of 9 for a period of about 6 minutes.The spectrophotometric determination is then carried out.

Recently, it was discovered that LDH is comprised of five isoenzymes,designated for the sake of simplicity as LDH LDH LDH LDH,,, and LDHThese isoenzymes have been separated and identified by electrophoresis.

The importance in knowing of the existence of these isoenzymes ismanifested in its application as a diagnostic tool. More particularly,the proportion of different isoenzyme fractions (i.e., the isoenzymepattern) varies from tissue to tissue. For instance, cardiac extractsexhibit a pattern where fractions LDH and LDH predominate.

Correspondingly, hepatic tissue shows a preponderance of the LDHfraction.

The usefulness of these observations can be applied practically since anincrease in normal serum LDH levels is ascribable to an increase in oneor more isoenzyme fractions which in turn may be traced to the affectedtissue.

For example, after myocardial infarction, the LDH level increases in theserum even before the total LDH activity exceeds normal limits and thismay facilitate early diagnosis. Furthermore, whereas total LDH activityremains elevated for an average of ten days after infarction, the LDHisoenzyme elevation commonly persists into the third week.

Another ofthe isoenzymes, namely LDH is found to increase in activity inacute and chronic liver disorders.

The most usual procedure for separation of the LDH isoenzymes iselectrophoresis and is still regarded as the standard in suchmeasurements. However, for practical use, it is unacceptable because ofthe time and labor involved.

Another method practiced in the art relates to heat fractionation of LDHinto the isoenzyme components whereby certain LDH isoenzyme fractionssuch as LDH, and LDH,; are stable at temperatures as high as 65C.whereas fraction LDH is inactivated at 57C. and LDH 3 and 4 becomeinactivated somewhere between 57C. and 65C. Because of this heatstability differential, one can make comparisons of the heat-stable andheat-labile LDH isoenzyme fractions. In this manner, one is permitted toassess the relative amounts of various isoenzyme fractions found withinLDH.

The heat-labile LDH, fraction (destroyed by heating at 59C.) constitutesabout 10% of the total LDH activity in normal serum. Accordingly, anincrease in LDH activity greater than 30% of the total (usually areading of 3070%) provides good indication that an acute or chronicliver disorder may be present in the patient.

If the combined activity of the LDH, and LDH, fractions (heat-stable)comprises or more of the total LDH activity, heart disfunction ordisease is indicated.

It is apparent from the foregoing discussion that the test is based on acomparative ratio between the total LDH activity and the heat labile orheat stable fractions.

The prior art method of fractionating the LDH in the serum sampleconsisted of heating the undiluted serum sample at 56C, the temperatureat which the LDH fraction is inactivated, for a period of about 30minutes at a pH of about 7.0, the pH of the sample.

The obvious drawback of the procedure in highly so phisticatedcontinuous stream diagnostic instruments is apparent. Where hundreds ofsamples are to be tested, it would be impractical to heat each one for30 minutes after assaying for total LDH activity. Such restriction wouldpreclude application to a continuous stream apparatus.

The present invention is directed to the elimination of thesedisadvantages. If effectively permits the analyses of numerous sampleson continuous stream at a rate feasible for continuous stream apparatus,and just as important, allows for an immediate determination of LDHfractionated activity with only a simple intervening heating step.

The present invention therefore makes the heat fractionation techniquecommercially practicable.

SUMMARY OF THE INVENTION This invention relates to an effective methodof fractionating LDH in a serum sample which comprises heating saidsample for a period of from about 0.5 to about 5 minutes at atemperature of from about 55C to about 65C at a pH in the range from 7to l0 maintained by an aqueous buffer solution, said bufferconcentration having an ionic strength sufficient to substantiallyinactivate the isoenzyme molecules and subsequently quick-cooling theresulting mixture.

Preferred embodiments of the above-described process comprise:

a. A heating period of about 40 seconds;

b. A buffer consisting of a 0.67 M aqueous solution of aminomethylpropanol;

c. A mixture pH of about 9;

d. A mixing temperature of about 625C; and

e. A cooling period of 30 to 60 seconds.

DETAILED DESCRIPTION OF THE INVENTION The unique feature of the hereindisclosed method of heat fractionating LDH in a serum sample as a meansfor diagnosing certain body disorders is its ability to be adapted to acontinuous stream process whereby numerous samples are assayedconsecutively. After rapid on-line heat fractionation and cooling, thesample is in condition for residual LDH assay.

The end result is derived from a comparison between the total LDH assayand the LDH fractionation assay. Therefore, if these steps are carriedout consecutively or simultaneously on parallel dual channels without anundesirable waiting period, the overall process is not only a timesaver, it also lends itself to a continuous stream analyzer.

The present process provides the integration of these steps. Thisintegration is brought about by the rapid heating step (about 40seconds) and subsequent quickcooling step prior to LDH fractionationassay.

The total and fractionated LDH activity can be measured under the samepH and buffer conditions. An aliquot of serum is assayed for total LDHat a pH of 7 to 10 and about 37C. An identical aliquot is rapidly heatedin the same range from 55C to about 65C, preferably about 625C, cooledand assayed for LDH residual activity.

It turns out there are four critical variables in the herein disclosedprocess, all interrelated with one another. Accordingly, the optimumconditions for a fixed pH in the range of 7-10 must necessarily beempirically determined.

It is important to realize that the pH during fractionation and ensuingassay be the same as that of the total LDH assay.

For example, it is found that when the pH is set or fixed at 9, which ispreferred, a heating period of about 40 seconds at a temperature ofapproximately 6263C is mandated when a buffer comprising aminomethylpropanol is used. Moreover, a 0.67 M solution is necessary to provide aconcentration of buffer having an ionic strength sufficient tosubstantially inactivate certain or specific isoenzyme molecules.

If the pH is less than 9, the heating step may require a longer timeperiod. It is possible that under certain conditions, the heating stepmay take as long as 10 or 15 minutes. Of course, it must be recognizedthat beyond about minutes the heating step will be of very little valueif a continuous step process is to be used.

correspondingly, if the pH is above 9 the heating time will beshortened. However beyond a pH of 10, the fractionation proceeds sorapidly as to be impractical. Moreover, adverse chemical reactions takeplace which result in misleading assays.

Since the mixture during heating must be restricted to a pH in the rangeof 7-10, and preferably 9, numerous buffer compositions become readilyapparent to one skilled in the art.

For instance, the following list, by no means exhaustive, enumeratesrepresentative buffers suitable for inclusion into the subject process:

tris (hydroxymethyl) aminomethane glycine triethanolamineborate-containing buffer ammonium-containing buffer Each of these buffercompositions is capable of maintaining a pH value in the range of 7-10.Once the pH is set, the other variables can then be determined toprovide the optimum conditions for that buffer and pH level. Thisdetermination is not complicated and the ultimately arrived atconditions will fall within the boundaries circumscribed herein.

Accordingly, one skilled in the art can choose beforehand each of thevariables at random provided they fall within the confines of theinvention as described. However, to determine the optimum set ofconditions. sim' 5 ple experimentation is necessary.

In sum, the unique advantages offered by the process of this inventioninclude:

1. suitable application to a continuous stream analyzer; and 2.elimination of a costly and time-consuming prior art procedurepreviously in use.

EXAMPLE I EXAMPLE ll The procedure of Example I is repeated on a serumsample from a patient with mycardial infarct. Electrophoretic analysisshowed elevated LDH l and LDH 2 isoenzymes. The fractionated LDH valuesaccording to the hereindisclosed procedure was greater than 85%.

These results indicate an agreement between the process of thisinvention and the standard electrophoretic method.

EXAMPLE Ill The procedure of Example I is repeated. On a serum sampletaken from a patient having liver disease. Electrophoretic analysisshowed elevated LDH 4 and 5. Analysis by the herein described procedureshowed a fractionated LDH value of 50%.

These results indicated an agreement between the process of thisinvention and the standard electrophoretic method.

EXAMPLE IV The procedure of Example l is substantially repeated usingthe following buffers in lieu of aminomethyl propanol:

tris (hydroxymethyl) aminomethane glycine triethanolamineborate-containing buffer quaternary ammonium salt-containing bufferEXAMPLE V The procedure of Example I is substantially repeated using thefollowing heating time periods with comparable results:

20 seconds 60 seconds 3 minutes EXAMPLE V1 The procedure of Example I issubstantially repeated at the following pH levels with comparableresults:

What is claimed is:

l. A method for effectively determining LDH activity in a serum sampleso as to diagnostically determine the existence and presence of diseasedorgans, comprising: mixing the serum sample with an aqueous buffersolution heating said serum sample-buffer solution mixture for a periodof from 0.5 to about 5 minutes at a pH in the range from 7.6 to at atemperature sufficient to fractionate the LDH, said aqueous buffersolution having an ionic strength sufficient to substantially inactivateparticular isoenzyme molecules during heating, rapidly cooling the serumsample-buffer solution to a temperature at least below 37C, and assayingsaid cooled sample for residual LDH activity at substantially said pH.

2. The method according to claim 1 wherein prior to said heating step,an identical aliquot of the sample is colorimetrically assayed for totalLDH activity.

3. The method according to claim 1 wherein said heating step is carriedout at a temperature between about 55C. and 65C.

4. The method according to claim 3 wherein said temperature is about625C.

5. The method according to claim 1 wherein said heating period is 40seconds.

6. The method according to claim 1 wherein said pH is 9.

7. The method according to claim 1 wherein said buffer is aminomethylpropanol in the form of an 0.67 M aqueous solution.

8. The method according to claim 1 wherein said buffer solution isaqueous tris (hydroxymethyl) aminomethane.

9. The method according to claim 1 wherein said buffer solution isaqueous glycine.

10. The method according to claim 1 wherein said buffer solution isaqueous triethanolamine.

11. The method according to claim 1 wherein said buffer solution is aborate-containing aqueous solution.

12. The method according to claim I wherein said buffer solution is aquarternary ammonium saltcontaining aqueous solution.

13. The method according to claim 1 wherein said cooling is accomplishedover a period of from 15 to 90 seconds.

14. A method of effectively determining LDH activity in a serum sampleto diagnostically determine the existence and presence of diseasedorgans which comprises heating said sample for a period of from 0.5 toabout 5 minutes at a temperature of from about 55C. to about C. at a pHin the range from 7.6 to H) maintained by an aqueous buffer solution,said buffer concentration having an ionic strength sufficient tosubstantially inactivate the isoenzyme molecules, followed by cooling ofthe resulting mixture to a temperature at least below 37C., and assayingsaid cooled sample for residual LDH activity at substantially said given15. The method according to claim 14 wherein said fractionation iseffected within a period of about 40 seconds.

16. The method according to claim 14 wherein said pH duringfractionation is about pH 9.

17. The method according to claim 14 wherein said buffer is aminomethylpropanol in the form of an 0.67M aqueous solution.

18. The method according to claim 14 wherein said fractionationtemperature is about 625C.

19. The method according to claim 14 wherein said buffered solution isaqueous tris (hydroxymethyl) aminomethane.

20. The method according to claim 14 wherein said buffer solution isaqueous glycine.

21. The method according to claim 14 wherein said buffer solution isaqueous triethanolamine.

22. The method according to claim 14 wherein said buffer solution is aborate-containing aqueous solution.

23. The method according to claim 14 wherein said buffer solution is aquaternary ammonium salt containing aqueous solution.

24. The method according to claim 14 wherein said cooling isaccomplished over a period of from 15 to

2. The method according to claim 1 wherein prior to said heating step, an identical aliquot of the sample is colorimetrically assayed for total LDH activity.
 3. The method according to claim 1 wherein said heating step is carried out at a temperature between about 55*C. and 65*C.
 4. The method according to claim 3 wherein said temperature is about 62.5*C.
 5. The method according to claim 1 wherein said heating period is 40 seconds.
 6. The method according to claim 1 wherein said pH is
 9. 7. The method according to claim 1 wherein said buffer is aminomethyl propanol in the form of an 0.67 M aqueous solution.
 8. The method according to claim 1 wherein said buffer solution is aqueous tris (hydroxymethyl) aminomethane.
 9. The method according to claim 1 wherein said buffer solution is aqueous glycine.
 10. The method according to claim 1 wherein said buffer solution is aqueous triethanolamine.
 11. The method according to claim 1 wherein said buffer solution is a borate-containing aqueous solution.
 12. The method according to claim 1 wherein said buffer solution is a quarternary ammonIum salt-containing aqueous solution.
 13. The method according to claim 1 wherein said cooling is accomplished over a period of from 15 to 90 seconds.
 14. A method of effectively determining LDH activity in a serum sample to diagnostically determine the existence and presence of diseased organs which comprises heating said sample for a period of from 0.5 to about 5 minutes at a temperature of from about 55*C. to about 65*C. at a pH in the range from 7.6 to 10 maintained by an aqueous buffer solution, said buffer concentration having an ionic strength sufficient to substantially inactivate the isoenzyme molecules, followed by cooling of the resulting mixture to a temperature at least below 37*C., and assaying said cooled sample for residual LDH activity at substantially said given pH.
 15. The method according to claim 14 wherein said fractionation is effected within a period of about 40 seconds.
 16. The method according to claim 14 wherein said pH during fractionation is about pH
 9. 17. The method according to claim 14 wherein said buffer is aminomethyl propanol in the form of an 0.67M aqueous solution.
 18. The method according to claim 14 wherein said fractionation temperature is about 62.5*C.
 19. The method according to claim 14 wherein said buffered solution is aqueous tris (hydroxymethyl) aminomethane.
 20. The method according to claim 14 wherein said buffer solution is aqueous glycine.
 21. The method according to claim 14 wherein said buffer solution is aqueous triethanolamine.
 22. The method according to claim 14 wherein said buffer solution is a borate-containing aqueous solution.
 23. The method according to claim 14 wherein said buffer solution is a quaternary ammonium salt containing aqueous solution.
 24. The method according to claim 14 wherein said cooling is accomplished over a period of from 15 to 90 seconds. 